PMC:3951193 / 56808-83531 JSONTXT

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modifications\n\nDiet and weight change\nDozens of animal studies and human cohort studies have shown that diets rich in fats and sugars alter levels of AEA, 2-AG, their metabolic enzymes, and CB1. The reverse causality is also true—many studies show that CB1 agonists stimulate the consumption of fat and sugar. The rewarding properties of palatable foods are attenuated by CB1 blockade and in CB1 −/− knockouts. Stimulation of feeding behavior by CB1 agonists occurs across the phylogenetic scale, from humans to Hydra, although there is no molecular evidence for CB1 orthologs in invertebrates other than the boneless chordates Ciona intestinalis and Branchiostoma floridae. Reviews on this topic are available [7], [228], [229], which we do not intend to duplicate here.\nUpregulation of the eCB system in obese humans seems to be driven by excessive production of eCBs in several peripheral tissues such as visceral adipose tissue, liver, pancreas, and skeletal muscle. Differences arise between central (intra-abdominal) adipocytes versus peripheral (subcutaneous) adipocytes, with additional variations due to gender, age, and genetic polymorphisms in metabolic enzymes. Visceral adiposity particularly correlates with elevated levels of 2-AG in blood plasma [230]. Increases in circulating eCBs likely reflect spillover from adipose tissues and liver parenchyma, where CB1 activation promotes de novo lipogenesis and reduces insulin sensitivity, respectively. In mice with diet-induced obesity, CB1 mRNA and protein levels increased in the hippocampus, compared to lean controls [231]. Furthermore, hippocampal slices from obese mice showed increased CB1 functionality, with no sign of CB1 desensitization. We find it surprising that sustained elevations of eCB ligands do not result in CB1 downregulation. This may be due to the fact that such elevations are not as dramatic as those caused, for example, by chronic MAGL inhibition. The lack of downregulation may contribute to the hedonic aspects of overeating, and influence cognitive processes.\nWeight loss by caloric restriction or fasting predictably modulates the eCB system. Animal studies have demonstrated the complexities arising in adipose tissue versus the central nervous system (Table 2). In human studies, weight loss from caloric restriction has produced conflicting results. Engeli et al. [232] measured CB1 and FAAH gene expression, and serum AEA and 2-AG, in obese postmenopausal women. They reported no changes after 5% weight loss from caloric restriction. Bennetzen et al. [233] analyzed a younger population of obese men and women; a 10–12% weight loss resulted in elevated 2-AG levels in gluteal adipose tissues, with no change in AEA levels. Weight loss increased CB1 mRNA in abdominal adipose tissues but decreased CB1 mRNA in gluteal adipose tissues.\n10.1371/journal.pone.0089566.t002 Table 2 Effects of short- and long-term caloric restriction upon the brain eCB system in animal studies. In centrally obese men, decreased plasma AEA and 2-AG levels accompanied a weight loss intervention consisting of both caloric restriction and exercise. Only 2-AG levels correlated with decreased visceral adipose tissue, plasma triglycerides and insulin resistance, and improved HDL-cholesterol levels [234]. However, the influence of caloric restriction and exercise separately was not analyzed in this study. You et al. [235] measured CB1 and FAAH mRNA in subcutaneous abdominal and gluteal adipose tissue in overweight or obese postmenopausal women. Caloric restriction resulted in 11% weight loss, which led to a reduction in gluteal CB1 and FAAH gene expression but no significant changes in abdominal adipose tissue. You and associates also tested the effects of exercise, see below. A 12-week hospital-based weight loss program (moderate caloric restriction along with counseling by dieticians and physical activity teachers) resulted in a mean weight loss of 9.5% and a significant reduction in salivary AEA levels, while salivary 2-AG, OEA and PEA did not significantly change [236].\nIn summary, increased food intake, adiposity, and elevated levels of AEA and 2-AG apparently spiral in a feed-forward mechanism. Weight loss from caloric restriction breaks the cycle, possibly by reducing CB1 expression and reducing eCB levels.\n\nExercise\nRodent studies have shown that exercise modulates the eCB system (Table 3). The results of these studies show a critical difference between short-term, voluntary exercises (e.g., wheel running) and long-term, coerced exercise (forced swimming, treadmills). Although both types of exercise regimens increased eCB ligand concentrations, only long-term-forced exercise led to sustained elevations of eCBs, and predictable CB1 downregulation.\n10.1371/journal.pone.0089566.t003 Table 3 Effects of exercise upon the eCB system in rodent studies. In humans, serum AEA levels doubled over baseline in male subjects after ≥30 min running, and increased significantly in male subjects after biking. Serum 2-AG levels did not significantly increase [237]. Heyman et al. [238] reported similar findings in male cyclists—serum AEA levels increased significantly during exercise, whereas 2-AG concentrations remained stable. AEA levels increased incrementally at 55% maximum work output, at 75% Wmax, and during a 15 min recovery period. Beta-endorphin levels exhibited a different trajectory—they did not increase until the 75% Wmax stage, and dropped significantly during the recovery period.\nFeuerecker et al. [239] measured the effects of physical exercise in aerobically-trained male subjects. Strenuous hiking at high altitudes (up to 3196 m) significantly increased serum AEA levels over baseline. Strenuous hiking at low altitudes also increased AEA level, but to a lesser extent. In a small cadre of overweight or obese middle-aged women, 20 weeks of moderate-intensity aerobic exercise (CRM) or vigorous-intensity aerobic exercise (CRV) did not change CB1 or FAAH gene expression [235]. However, combining data from the two groups (CRM+CRV) showed a decrease in FAAH mRNA in abdominal adipose tissue, compared to a control group that participated solely in caloric restriction. The CRM and CRV groups showed a slight increase in CB1 mRNA expression in gluteal adipose tissue over baseline, whereas the control group that only participated in caloric restriction showed a significant decrease in CB1 mRNA.\nRaichlen et al., [240] measured circulating eCBs in humans and dogs (cursorial mammals) and ferrets (a non-cursorial mammal) before and after treadmill exercise to test the hypothesis that neurobiological rewards are linked to high-intensity exercise in cursorial mammals. The authors showed that humans and dogs share significantly increased exercise-induced eCB signaling following high-intensity endurance running, whereas eCB signaling did not significantly increase following low-intensity walking, nor did it increase in the non-cursorial ferrets following exercise at any intensity. The same research group showed that serum AEA levels in male and female runners significantly increased after 30 minutes of moderately intense treadmill running (70–80% age-adjusted maximum heart rate), and not after very high or very low intensity exercises [240], [241].\nIn summary, medium- to high-intensity voluntary exercise in cursorial mammals, including humans, increases eCB signaling, via increased serum AEA levels (but not 2-AG), and possibly increased CB1 expression. “Runner's high” may be an eCB-induced reward for exercise.\n\nAlcohol\nAcute administration of a high dose of ethanol in rats decreased AEA levels in brain, serum, and adipose tissue; PEA also decreased in the brain. AEA decrease was associated with inhibition of AEA release and no change in NAPE-PLD or FAAH hydrolysis [242]. However, exposing ex vivo murine hippocampal neuron cultures to lower doses of ethanol increased AEA and 2-AG release [243]. This increase led to reduced presynaptic glutamate release in neuron cultures, which was blocked by SR141716A. There was no change in CB1 density.\nElectrophysiological studies of anesthetized rats showed that alcohol enhanced eCB signaling in mesolimbic circuits [244]. This effect was blocked by SR141716A, and increased by the FAAH inhibitor URB597—indicating AEA involvement. Another study by the same group showed parallel responses in rat amygdala. The downregulation of amygdala CB1 with chronic WIN55212-2 blunted the response to alcohol [245].\nEx vivo exposure of rat striatal slices showed ethanol shifts synaptic plasticity from LTP to eCB-mediated LTDI. Ethanol-enhanced LTDI was blocked by the CB1 antagonist AM251 [246]. The same group showed that ethanol modulated eCB-mediated striatal plasticity in a synapse-specific manner. Ethanol prevented CB1-dependent long-lasting disinhibition (DLL) in the dorsolateral striatum [247]. Furthermore, the study showed that LTDI by an exogenous cannabinoid, WIN55,212-2, was actually prevented by ethanol.\nChronic ethanol treatment decreased CB1 density and decreased cannabinoid-stimulated [35S]GTPγS activation in various animal models [248]–[251]. One study of chronic ethanol did not alter CB1 binding of [3H]CP55,940 or CB1 mRNA levels in rat brain homogenates [68]. Short-term chronic exposure (72 hours) of ethanol vapor in mice increased CB1 density in the cortex, hippocampus, striatum and cerebellum, with downregulation of CB1 receptor-stimulated [35S]GTPγS binding [252]. The effects of chronic ethanol treatment upon eCB levels in various in vitro and animal models are shown in Table 4.\n10.1371/journal.pone.0089566.t004 Table 4 Effects of chronic or subchronic ethanol upon eCB levels. 1 ↑, increase; ↓, decrease; ≈, no change; assay; result compared to unsupplemented controls. Vinod et al. [251] compared alcohol-preferring (aP) and alcohol-non preferring (NaP) rats, a pair of rat lines selectively bred for opposite alcohol preference. CB1 receptor density, CB1 receptor-stimulated [35S]GTPγS coupling, and levels of AEA and 2-AG were higher in the brains of alcohol-naive aP compared to NaP rats. Ethanol consumption in aP rats decreased CB1 receptor-stimulated [35S]GTPγS binding after 10 days, and moreso after 60 days. 2-AG levels elevated after 10 days, and both 2-AG and AEA levels increased after 60 days; FAAH levels decreased with no change in MAGL. Ethanol withdrawl upregulated [35S]GTPγS binding.\nA rat model of binge drinking—serial cycles of ethanol intoxication and withdrawal—increased CB1 mRNA in the prefrontal cortex [253]. Another study of serial cycles in rats showed a transient decrease in hippocampal CB1 mRNA and protein levels (two days after cessation of cycles), followed by a long term up-regulation in CB1 mRNA and protein, 40 days after cessation of cycles. Serial cycles increased 2-AG in the hippocampus, two days and 40 days after cessation of cycles; AEA increased only at 40 days [254].\nAn electrophysiological study of intermittent ethanol consumption in rats showed depression of CB1-dependent long-lasting disinhibition (DLL) in excised slices of the dorsolateral striatum [255]. Furthermore, the study showed that LTDI by an exogenous cannabinoid, WIN55,212-2, was prevented by intermittent ethanol consumption.\nA human clinical trial assigned 55 adults to one of three groups—drinking either 250 ml of red wine, grape juice, or plain water. Within 10 minutes, the consumption of a moderate amount of alcohol reduces plasma AEA and 2-AG concentrations, whereas an equal volume of grape juice did not affect plasma eCBs. Interestingly, plain water reduced 2-AG concentrations without affecting AEA [256].\nAlcoholics who died of natural causes or motor vehicle accidents expressed decreased CB1 densities in the ventral striatum, decreased CP55,940-stimulated [35S]GTPγS binding, and decreased FAAH activity, compared to controls [257]. Alcoholics who died of suicide in the same study had increased CB1 densities, increased CB1 receptor-stimulated G(i/o) protein activation, and decreased FAAH activity, compared to controls. Lehtonen et al. (2010) measured eCB levels in post-mortem brains of Cloninger type 1 and type 2 alcoholics. Type 1 alcoholics had lower levels of AEA than controls in the nucleus accumbens (NAcc), anterior cingulate cortex, and frontal cortex. PEA, OEA, and 2-AG were unchanged. They also showed dopaminergic deficiencies in the NAcc, suggesting a compensatory mechanism one direction or the other. Type 2 alcoholics produced slightly higher eCB levels than controls, but not significantly.\nIn summary, acute ethanol may enhance endogenous eCB release and eCB signaling, although it varies by brain area and synapse, and this complexity requires further testing. Two studies suggest ethanol dampens the effects of the eCB system. Chronic ethanol consumption and binge drinking likely desensitize or downregulate CB1 and impair eCB signaling, except perhaps in areas involved in reward and motivation to self-administer this substance of abuse [258].\n\nNicotine\nIn a human randomized controlled trial, nicotine augmented THC-induced “high” and heart rate [259]. In rodent behavioral studies, acute nicotine augmented THC discrimination and THC-induced hypothermia, antinociception, locomotor inactivity, anxiolysis, and place aversion [260]–[264]. Nicotine-potentiated THC discrimination was blocked by rimonabant and URB-597 (a FAAH inhibitor), suggesting nicotine potentiation is mediated by the release of AEA acting at CB1 [263]. CB2 is also involved—the CB2–selective agonist JWH133 induced antinociception in the mouse formalin test, and this effect was potentiated by nicotine [265]. Acute nicotine elicited marked increases in AEA in the amygdala, hypothalamus, and prefrontal cortex but decreased levels in the hippocampus; variations in 2-AG were less pronounced [266].\nIn a contrary study, intracelebellar microinfusion of nicotine attenuated THC-induced ataxia in mice. Microinfusion of synthetic subtype agonists indicated the involvement of α4β2 but not α7 nicotinic receptor subtypes [267]. Buczynski et al. [268] compared volitional self-administration (SA) versus forced nicotine exposure (FA) in the ventral tegmental area using in vivo microdialysis. SA but not FA increased AEA; both SA and FA increased 2-AG; these subtle changes were not seen in corresponding bulk brain tissue analysis of eCBs. Acute nicotine enhanced THC-induced c-Fos expression in various brain regions [264].\nChronic nicotine increased AEA levels in the limbic forebrain and increased AEA and 2-AG contents in rat brainstem, but decreased AEA and/or 2-AG contents in the hippocampus, the striatum and the cerebral cortex [258]. Chronic nicotine increased CB1 density in the prelimbic prefrontal cortex, ventral tegmental area, and the hippocampus [269]. Seven days of nicotine exposure increased brain CB1 densities in adolescent male rats and sensitized them to the locomotor-decreasing effects of THC and CP55,940 [270]. These changes were not seen in adult male rats. Chronic nicotine inhibited the development of tolerance to antinociceptive and hypothermic effects of THC [264].\nOther plant products that exert cholinergic effects, such as calamus, Acorus calamus, have been admixed with cannabis to decrease cannabis-induced memory deficits, and “calm and center the effects of marijuana” [42]. Consistent with this, the synthetic cholinergic agent rivastigmine reversed memory deficits in rats induced by the synthetic cannabinoid WIN55,212-2 [271].\n\nCaffeine\nCo-administering caffeine and cannabis has a long history. Bell [272] claimed that oral administration of hashish with coffee increased the effects of cannabis, and at the same time diminished its duration. He proposed a pharmacokinetic mechanism—coffee promoted more rapid absorption of hashish.\nCaffeine and theophylline are antagonists of adenosine receptors. Adenosine receptors are tonically activated by adenosine, their endogenous ligand. Rodent studies indicate that A1-subtype adenosine receptors tonically inhibit CB1 activity [273]. Thus the antagonism of A1 receptors by caffeine and theophylline enhances eCB system function (e.g., activation of CB1 by 2-AG). Caffeine potentiated CB1-mediated activity stimulated by THC and WIN-55,212 in hippocampus slices [273]. Consistent with this, the simultaneous application of WIN-55,212 plus an A1 agonist produced less than additive stimulation of [35S]GTPγS binding in mouse cerebellar membranes [274].\nIn whole animals, however, caffeine's effects are biphasic and vary by dosage and acute versus chronic administration. In humans, the acute administration of caffeine decreases headache pain, but exposure to chronic high doses, ≥300 mg/day, may exacerbate chronic pain [275]. In rabbits, an acute dose of caffeine antagonized THC-induced changes in cortico-hippocampal electroencephalogram recordings [276]. In mice, chronic caffeine at high doses potentiated CB1-dependent stimulation by eCBs and HU210 at striatal GABAergic, but not glutamatergic, synapses [277]. A single dose or a subacute dose (one day of caffeine in water) rescued the sensitivity of GABAergic synapses to HU210 in mice exposed to chronic stress.\nChronic caffeine at moderate doses increased THC's effects on short-term memory in mice [278]. Surprisingly, CB1 density decreased in the caffeinated mice, measured by [3H]SR141716A binding. Cortical and hippocampal tissues also showed a decrease in WIN55,212-2-stimulated [35S]GTPγS binding, but this attenuation was not seen in THC-stimulated [35S]GTPγS binding. This highlights the fact that caffeine-induced changes observed in vitro do not necessarily reflect the effects of caffeine upon integrated brain circuitry in vivo. Lastly, acute antagonism of A1 with DPCPX did not modulate the effects of THC on short-term memory [278], which further supports our hypothesis that chronic and acute blockade of A1 receptors have different functional consequences.\n\nCannabis\nCannabis and cannabis products are complex polypharmaceuticals, consisting of THC, cannabidiol (CBD), dozens of minor cannabinoids, as well as terpenoids, flavonoids, and other compounds. Fundamentally, THC mimics AEA and 2-AG by acting as an agonist at CB1 and CB2 [279]. But rather than simply substituting for AEA and 2-AG, McPartland and Guy [280] proposed that Cannabis and its many constituents work, in part, by “kick-starting” the eCB system. The acute administration of THC increased CB1 density in rodent brains [281], [282]. Acute upregulation of CB1 mRNA continued for up to 14 days in some rat brain regions [283]. Acute THC also increased the sensitivity of CB1 to cannabinoids, measured by WIN-55,212-2-stimulated [35S]GTPγS binding in rat brains [284]. Lastly, acute THC stimulated AEA biosynthesis [285].\nChronic, high dosing of THC causes a predictable desensitization and downregulation of CB1 and CB2, accompanied by drug tolerance. Chronic THC decreased CB1 density in rodent brains, and dampened cannabinoid-stimulated [35S]GTPγS [282], [284], [286], [287]. CB1 in different regions of the brain downregulate and desensitize at unequal rates and magnitudes, with greatest decreases in the hippocampus and little or no change in the nucleus accumbens and basolateral amygdala. Chronic THC elicited few changes in AEA or 2-AG levels in rat brains, except for a significant augmentation of AEA levels in the limbic forebrain [288].\nSimilar results have been reported in two human studies. Villares [289] collected postmortem brain tissues from known cannabis smokers; [3H]SR141716A binding and CB1 mRNA was downregulated in several brain regions, compared to non-smoking control autopsies. Hirvonen et al. [290] employed PET scan imaging in living subjects. The degree of CB1 downregulation correlated with years of chronic cannabis smoking. CB1 densities returned to normal after four weeks of abstinence. Variable downregulation in different brain regions may explain why frequent users of cannabis develop tolerance to some effects of THC, such as anxiogenesis and cognitive impairment, but not to its euphoric effects [291]. Downregulation is partially epigenetic—the CB1 promoter region in chronic marijuana smokers is hypermethylated, reducing CB1 mRNA expression levels [292].\nTHC acts as a partial agonist of CB1, compared to synthetic cannabinoids which act as full agonists (Table 5). Partial agonism likely explains why exposure to THC caused half as much CB1 desensitization as the full agonist WIN55,212-2 in rat hippocampal neurons [293]. In a study of rat CB1 transfected into AtT20 cells, THC caused less downregulation and internalization than WIN55,212-2 or CP-55,940 [294]. In agreement, drug tolerance studies utilizing the behavioral “tetrad” test show that chronic THC caused less tolerance than the full agonist CP-55,940 in mice [295]. In a study of human CB1 transfected into Xenopus oocytes, the desensitization rate of THC was half that of WIN55,212-2 [296]. However, one [35S]GTPγS autoradiography study of rat brains suggested that chronic THC and WIN55,212-2 caused equal desensitization [297]. Another study indicated that THC acts as a full agonist at mouse GABAergic synapses, with efficacy equal to WIN55,212-2, albeit at fairly high concentrations [298].\n10.1371/journal.pone.0089566.t005 Table 5 Partial agonism of THC at CB1, based on assays of cannabinoid-stimulated signal transduction. If THC is a partial agonist, then THC might functionally antagonize the effects of a full agonist when the two drugs are added together. THC antagonized the effects of WIN55,212-2 in rat brain sections [284], [299], and mouse autaptic hippocampal neurons [300].\nThe capacity of THC to antagonize a full agonist depends, in part, upon ligand affinity—its ability to occupy and hold the CB1 binding site. A meta-analysis of affinity studies calculated a mean Ki = 42.6 nM for THC in rat membranes—much less affinity than that of WIN-55,940, with a Kd = 2.4 nM [22]. This indicates that high concentrations of THC relative to WIN-55,940 are required to antagonize the full agonist. There are species differences—in human membranes, CB1 affinity of THC (Ki = 25.1 nM) is much closer to that of WIN-55,940 (Kd = 16.7).\n2-AG acts as a full agonist at rodent and human CB1 and CB2 [296], [301]–[303]. The emetogenic effects of exogenously-administered 2-AG were blocked by THC [304]. THC dampened or occluded eCB-mediated retrograde signaling of CB1, presumable mediated by 2-AG [300], [305], [306]. Roloff and Thayer [307] demonstrated another complexity in the relationship between THC and 2-AG: neuron firing rate in response to stimulus in rat hippocampal neurons. At low firing rates, THC mimicked 2-AG and behaved like an agonist; at high firing rates, THC antagonized endogenous 2-AG signaling.\nAEA is a partial agonist like THC, with an efficacy somewhat greater than THC in mouse brain [308] and transfected human CB1 [296]. Consistent with partial agonism, exogenously-administered AEA caused little tolerance in rodents [309], [310]. Agonist trafficking adds further complexity—THC and AEA preferentially activate different G-protein subtypes [311]. At transfected human CB1, AEA acted as a full agonist via Gαi subunits, and a partial agonist via Gαo subunits, with agonist efficacy much greater than THC at Gαi, and slightly greater than THC at Gαo [312].\nAEA and THC can antagonize each other; this in part is due to cross-tolerance [313], [314]. Falenski et al. [287] demonstrated that subchronic administration of THC in FAAH−/− knockout mice caused greater tolerance to THC than did subchronic administration of THC in wildtype mice. Thus elevated levels of AEA in FAAH−/− knockouts produced additive effects with THC. Vann et al. [315] trained rats to discriminate THC; trained rats injected with PMSF, which inhibits FAAH, showed 2.7-fold greater discrimination than rats injected with vehicle. In other words, inhibiting AEA degradation led to an increase in the potency of THC. Further, THC was more potent at producing antinociception, decreasing spontaneous activity, and increasing ring immobility when co-administered with PMSF as compared to vehicle.\nIn summary, the effects of THC upon the eCB system oscillate between potentiation and suppression, depending on acute versus chronic dosage. The dividing line between “acute” and “chronic” is a gray zone, and likely differs amongst individuals. Suplita et al. [316] summarized the situation: they studied “stress antinociception,” where rodents become less responsive to painful stimuli following exposure to an environmental stressor. Stress antinociception is mediated, in part, by the coordinated release of 2-AG and AEA. Acute administration of THC potentiated eCB-mediated stress antinociception. The converse was also true: animals exposed acutely to foot shock, which elicits eCB-mediated stress antinociception, became sensitized to the effects of THC. Chronic administration of THC predictably dampened stress antinociception. The converse was not true: chronic exposure to foot shock (3 min/day for 15 days) failed to dampen antinociception induced by either WIN-55,212-2 or by further footshocks.\nThe potential synergy between THC and the eCB system is analogous to the potential synergy between AEA and 2-AG: Rodent studies that combined FAAH and MAGL inhibitors indicated that AEA and 2-AG may activate CB1 receptors in different parts of the central nervous system. Each causes unique behavioral effects, and when both are enhanced, new effects emerge. Long and colleagues [317] showed that AEA and 2-AG independently dampen pain sensation, but together their effects are dramatically enhanced.\nCannabis is more than THC [318], [319]. Adding CBD to THC in mice enhanced CB1 expression in hippocampus and hypothalamus [320]. CBD increased hippocampal cell survival and neurogenesis, whereas THC had the opposite effect; the CBD response was absent in CB1 −/− knockout mice [321]. CBD inhibited the cellular uptake of AEA and its breakdown by FAAH [322], [323]. A separate systematic review regarding the effects of CBD on THC is currently underway (McPartland, unpublished). Several other non-THC cannabinoids interact with enzymes of the eCB system. For example, cannabidivarin and cannabidiolic acid are moderately potent inhibitors of DAGLα, and cannabigerol and cannabichromene are relatively potent inhibitors of anandamide cellular uptake [323]. Interestingly, cannabis extracts (“botanical drug substances,” BDS) enriched in cannabinoids, such as THC-acid BDS and CBD-BDS, were more potent than the corresponding pure compounds at inhibiting MAGL and AEA cellular uptake [323]."}

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modifications\n\nDiet and weight change\nDozens of animal studies and human cohort studies have shown that diets rich in fats and sugars alter levels of AEA, 2-AG, their metabolic enzymes, and CB1. The reverse causality is also true—many studies show that CB1 agonists stimulate the consumption of fat and sugar. The rewarding properties of palatable foods are attenuated by CB1 blockade and in CB1 −/− knockouts. Stimulation of feeding behavior by CB1 agonists occurs across the phylogenetic scale, from humans to Hydra, although there is no molecular evidence for CB1 orthologs in invertebrates other than the boneless chordates Ciona intestinalis and Branchiostoma floridae. Reviews on this topic are available [7], [228], [229], which we do not intend to duplicate here.\nUpregulation of the eCB system in obese humans seems to be driven by excessive production of eCBs in several peripheral tissues such as visceral adipose tissue, liver, pancreas, and skeletal muscle. Differences arise between central (intra-abdominal) adipocytes versus peripheral (subcutaneous) adipocytes, with additional variations due to gender, age, and genetic polymorphisms in metabolic enzymes. Visceral adiposity particularly correlates with elevated levels of 2-AG in blood plasma [230]. Increases in circulating eCBs likely reflect spillover from adipose tissues and liver parenchyma, where CB1 activation promotes de novo lipogenesis and reduces insulin sensitivity, respectively. In mice with diet-induced obesity, CB1 mRNA and protein levels increased in the hippocampus, compared to lean controls [231]. Furthermore, hippocampal slices from obese mice showed increased CB1 functionality, with no sign of CB1 desensitization. We find it surprising that sustained elevations of eCB ligands do not result in CB1 downregulation. This may be due to the fact that such elevations are not as dramatic as those caused, for example, by chronic MAGL inhibition. The lack of downregulation may contribute to the hedonic aspects of overeating, and influence cognitive processes.\nWeight loss by caloric restriction or fasting predictably modulates the eCB system. Animal studies have demonstrated the complexities arising in adipose tissue versus the central nervous system (Table 2). In human studies, weight loss from caloric restriction has produced conflicting results. Engeli et al. [232] measured CB1 and FAAH gene expression, and serum AEA and 2-AG, in obese postmenopausal women. They reported no changes after 5% weight loss from caloric restriction. Bennetzen et al. [233] analyzed a younger population of obese men and women; a 10–12% weight loss resulted in elevated 2-AG levels in gluteal adipose tissues, with no change in AEA levels. Weight loss increased CB1 mRNA in abdominal adipose tissues but decreased CB1 mRNA in gluteal adipose tissues.\n10.1371/journal.pone.0089566.t002 Table 2 Effects of short- and long-term caloric restriction upon the brain eCB system in animal studies. In centrally obese men, decreased plasma AEA and 2-AG levels accompanied a weight loss intervention consisting of both caloric restriction and exercise. Only 2-AG levels correlated with decreased visceral adipose tissue, plasma triglycerides and insulin resistance, and improved HDL-cholesterol levels [234]. However, the influence of caloric restriction and exercise separately was not analyzed in this study. You et al. [235] measured CB1 and FAAH mRNA in subcutaneous abdominal and gluteal adipose tissue in overweight or obese postmenopausal women. Caloric restriction resulted in 11% weight loss, which led to a reduction in gluteal CB1 and FAAH gene expression but no significant changes in abdominal adipose tissue. You and associates also tested the effects of exercise, see below. A 12-week hospital-based weight loss program (moderate caloric restriction along with counseling by dieticians and physical activity teachers) resulted in a mean weight loss of 9.5% and a significant reduction in salivary AEA levels, while salivary 2-AG, OEA and PEA did not significantly change [236].\nIn summary, increased food intake, adiposity, and elevated levels of AEA and 2-AG apparently spiral in a feed-forward mechanism. Weight loss from caloric restriction breaks the cycle, possibly by reducing CB1 expression and reducing eCB levels.\n\nExercise\nRodent studies have shown that exercise modulates the eCB system (Table 3). The results of these studies show a critical difference between short-term, voluntary exercises (e.g., wheel running) and long-term, coerced exercise (forced swimming, treadmills). Although both types of exercise regimens increased eCB ligand concentrations, only long-term-forced exercise led to sustained elevations of eCBs, and predictable CB1 downregulation.\n10.1371/journal.pone.0089566.t003 Table 3 Effects of exercise upon the eCB system in rodent studies. In humans, serum AEA levels doubled over baseline in male subjects after ≥30 min running, and increased significantly in male subjects after biking. Serum 2-AG levels did not significantly increase [237]. Heyman et al. [238] reported similar findings in male cyclists—serum AEA levels increased significantly during exercise, whereas 2-AG concentrations remained stable. AEA levels increased incrementally at 55% maximum work output, at 75% Wmax, and during a 15 min recovery period. Beta-endorphin levels exhibited a different trajectory—they did not increase until the 75% Wmax stage, and dropped significantly during the recovery period.\nFeuerecker et al. [239] measured the effects of physical exercise in aerobically-trained male subjects. Strenuous hiking at high altitudes (up to 3196 m) significantly increased serum AEA levels over baseline. Strenuous hiking at low altitudes also increased AEA level, but to a lesser extent. In a small cadre of overweight or obese middle-aged women, 20 weeks of moderate-intensity aerobic exercise (CRM) or vigorous-intensity aerobic exercise (CRV) did not change CB1 or FAAH gene expression [235]. However, combining data from the two groups (CRM+CRV) showed a decrease in FAAH mRNA in abdominal adipose tissue, compared to a control group that participated solely in caloric restriction. The CRM and CRV groups showed a slight increase in CB1 mRNA expression in gluteal adipose tissue over baseline, whereas the control group that only participated in caloric restriction showed a significant decrease in CB1 mRNA.\nRaichlen et al., [240] measured circulating eCBs in humans and dogs (cursorial mammals) and ferrets (a non-cursorial mammal) before and after treadmill exercise to test the hypothesis that neurobiological rewards are linked to high-intensity exercise in cursorial mammals. The authors showed that humans and dogs share significantly increased exercise-induced eCB signaling following high-intensity endurance running, whereas eCB signaling did not significantly increase following low-intensity walking, nor did it increase in the non-cursorial ferrets following exercise at any intensity. The same research group showed that serum AEA levels in male and female runners significantly increased after 30 minutes of moderately intense treadmill running (70–80% age-adjusted maximum heart rate), and not after very high or very low intensity exercises [240], [241].\nIn summary, medium- to high-intensity voluntary exercise in cursorial mammals, including humans, increases eCB signaling, via increased serum AEA levels (but not 2-AG), and possibly increased CB1 expression. “Runner's high” may be an eCB-induced reward for exercise.\n\nAlcohol\nAcute administration of a high dose of ethanol in rats decreased AEA levels in brain, serum, and adipose tissue; PEA also decreased in the brain. AEA decrease was associated with inhibition of AEA release and no change in NAPE-PLD or FAAH hydrolysis [242]. However, exposing ex vivo murine hippocampal neuron cultures to lower doses of ethanol increased AEA and 2-AG release [243]. This increase led to reduced presynaptic glutamate release in neuron cultures, which was blocked by SR141716A. There was no change in CB1 density.\nElectrophysiological studies of anesthetized rats showed that alcohol enhanced eCB signaling in mesolimbic circuits [244]. This effect was blocked by SR141716A, and increased by the FAAH inhibitor URB597—indicating AEA involvement. Another study by the same group showed parallel responses in rat amygdala. The downregulation of amygdala CB1 with chronic WIN55212-2 blunted the response to alcohol [245].\nEx vivo exposure of rat striatal slices showed ethanol shifts synaptic plasticity from LTP to eCB-mediated LTDI. Ethanol-enhanced LTDI was blocked by the CB1 antagonist AM251 [246]. The same group showed that ethanol modulated eCB-mediated striatal plasticity in a synapse-specific manner. Ethanol prevented CB1-dependent long-lasting disinhibition (DLL) in the dorsolateral striatum [247]. Furthermore, the study showed that LTDI by an exogenous cannabinoid, WIN55,212-2, was actually prevented by ethanol.\nChronic ethanol treatment decreased CB1 density and decreased cannabinoid-stimulated [35S]GTPγS activation in various animal models [248]–[251]. One study of chronic ethanol did not alter CB1 binding of [3H]CP55,940 or CB1 mRNA levels in rat brain homogenates [68]. Short-term chronic exposure (72 hours) of ethanol vapor in mice increased CB1 density in the cortex, hippocampus, striatum and cerebellum, with downregulation of CB1 receptor-stimulated [35S]GTPγS binding [252]. The effects of chronic ethanol treatment upon eCB levels in various in vitro and animal models are shown in Table 4.\n10.1371/journal.pone.0089566.t004 Table 4 Effects of chronic or subchronic ethanol upon eCB levels. 1 ↑, increase; ↓, decrease; ≈, no change; assay; result compared to unsupplemented controls. Vinod et al. [251] compared alcohol-preferring (aP) and alcohol-non preferring (NaP) rats, a pair of rat lines selectively bred for opposite alcohol preference. CB1 receptor density, CB1 receptor-stimulated [35S]GTPγS coupling, and levels of AEA and 2-AG were higher in the brains of alcohol-naive aP compared to NaP rats. Ethanol consumption in aP rats decreased CB1 receptor-stimulated [35S]GTPγS binding after 10 days, and moreso after 60 days. 2-AG levels elevated after 10 days, and both 2-AG and AEA levels increased after 60 days; FAAH levels decreased with no change in MAGL. Ethanol withdrawl upregulated [35S]GTPγS binding.\nA rat model of binge drinking—serial cycles of ethanol intoxication and withdrawal—increased CB1 mRNA in the prefrontal cortex [253]. Another study of serial cycles in rats showed a transient decrease in hippocampal CB1 mRNA and protein levels (two days after cessation of cycles), followed by a long term up-regulation in CB1 mRNA and protein, 40 days after cessation of cycles. Serial cycles increased 2-AG in the hippocampus, two days and 40 days after cessation of cycles; AEA increased only at 40 days [254].\nAn electrophysiological study of intermittent ethanol consumption in rats showed depression of CB1-dependent long-lasting disinhibition (DLL) in excised slices of the dorsolateral striatum [255]. Furthermore, the study showed that LTDI by an exogenous cannabinoid, WIN55,212-2, was prevented by intermittent ethanol consumption.\nA human clinical trial assigned 55 adults to one of three groups—drinking either 250 ml of red wine, grape juice, or plain water. Within 10 minutes, the consumption of a moderate amount of alcohol reduces plasma AEA and 2-AG concentrations, whereas an equal volume of grape juice did not affect plasma eCBs. Interestingly, plain water reduced 2-AG concentrations without affecting AEA [256].\nAlcoholics who died of natural causes or motor vehicle accidents expressed decreased CB1 densities in the ventral striatum, decreased CP55,940-stimulated [35S]GTPγS binding, and decreased FAAH activity, compared to controls [257]. Alcoholics who died of suicide in the same study had increased CB1 densities, increased CB1 receptor-stimulated G(i/o) protein activation, and decreased FAAH activity, compared to controls. Lehtonen et al. (2010) measured eCB levels in post-mortem brains of Cloninger type 1 and type 2 alcoholics. Type 1 alcoholics had lower levels of AEA than controls in the nucleus accumbens (NAcc), anterior cingulate cortex, and frontal cortex. PEA, OEA, and 2-AG were unchanged. They also showed dopaminergic deficiencies in the NAcc, suggesting a compensatory mechanism one direction or the other. Type 2 alcoholics produced slightly higher eCB levels than controls, but not significantly.\nIn summary, acute ethanol may enhance endogenous eCB release and eCB signaling, although it varies by brain area and synapse, and this complexity requires further testing. Two studies suggest ethanol dampens the effects of the eCB system. Chronic ethanol consumption and binge drinking likely desensitize or downregulate CB1 and impair eCB signaling, except perhaps in areas involved in reward and motivation to self-administer this substance of abuse [258].\n\nNicotine\nIn a human randomized controlled trial, nicotine augmented THC-induced “high” and heart rate [259]. In rodent behavioral studies, acute nicotine augmented THC discrimination and THC-induced hypothermia, antinociception, locomotor inactivity, anxiolysis, and place aversion [260]–[264]. Nicotine-potentiated THC discrimination was blocked by rimonabant and URB-597 (a FAAH inhibitor), suggesting nicotine potentiation is mediated by the release of AEA acting at CB1 [263]. CB2 is also involved—the CB2–selective agonist JWH133 induced antinociception in the mouse formalin test, and this effect was potentiated by nicotine [265]. Acute nicotine elicited marked increases in AEA in the amygdala, hypothalamus, and prefrontal cortex but decreased levels in the hippocampus; variations in 2-AG were less pronounced [266].\nIn a contrary study, intracelebellar microinfusion of nicotine attenuated THC-induced ataxia in mice. Microinfusion of synthetic subtype agonists indicated the involvement of α4β2 but not α7 nicotinic receptor subtypes [267]. Buczynski et al. [268] compared volitional self-administration (SA) versus forced nicotine exposure (FA) in the ventral tegmental area using in vivo microdialysis. SA but not FA increased AEA; both SA and FA increased 2-AG; these subtle changes were not seen in corresponding bulk brain tissue analysis of eCBs. Acute nicotine enhanced THC-induced c-Fos expression in various brain regions [264].\nChronic nicotine increased AEA levels in the limbic forebrain and increased AEA and 2-AG contents in rat brainstem, but decreased AEA and/or 2-AG contents in the hippocampus, the striatum and the cerebral cortex [258]. Chronic nicotine increased CB1 density in the prelimbic prefrontal cortex, ventral tegmental area, and the hippocampus [269]. Seven days of nicotine exposure increased brain CB1 densities in adolescent male rats and sensitized them to the locomotor-decreasing effects of THC and CP55,940 [270]. These changes were not seen in adult male rats. Chronic nicotine inhibited the development of tolerance to antinociceptive and hypothermic effects of THC [264].\nOther plant products that exert cholinergic effects, such as calamus, Acorus calamus, have been admixed with cannabis to decrease cannabis-induced memory deficits, and “calm and center the effects of marijuana” [42]. Consistent with this, the synthetic cholinergic agent rivastigmine reversed memory deficits in rats induced by the synthetic cannabinoid WIN55,212-2 [271].\n\nCaffeine\nCo-administering caffeine and cannabis has a long history. Bell [272] claimed that oral administration of hashish with coffee increased the effects of cannabis, and at the same time diminished its duration. He proposed a pharmacokinetic mechanism—coffee promoted more rapid absorption of hashish.\nCaffeine and theophylline are antagonists of adenosine receptors. Adenosine receptors are tonically activated by adenosine, their endogenous ligand. Rodent studies indicate that A1-subtype adenosine receptors tonically inhibit CB1 activity [273]. Thus the antagonism of A1 receptors by caffeine and theophylline enhances eCB system function (e.g., activation of CB1 by 2-AG). Caffeine potentiated CB1-mediated activity stimulated by THC and WIN-55,212 in hippocampus slices [273]. Consistent with this, the simultaneous application of WIN-55,212 plus an A1 agonist produced less than additive stimulation of [35S]GTPγS binding in mouse cerebellar membranes [274].\nIn whole animals, however, caffeine's effects are biphasic and vary by dosage and acute versus chronic administration. In humans, the acute administration of caffeine decreases headache pain, but exposure to chronic high doses, ≥300 mg/day, may exacerbate chronic pain [275]. In rabbits, an acute dose of caffeine antagonized THC-induced changes in cortico-hippocampal electroencephalogram recordings [276]. In mice, chronic caffeine at high doses potentiated CB1-dependent stimulation by eCBs and HU210 at striatal GABAergic, but not glutamatergic, synapses [277]. A single dose or a subacute dose (one day of caffeine in water) rescued the sensitivity of GABAergic synapses to HU210 in mice exposed to chronic stress.\nChronic caffeine at moderate doses increased THC's effects on short-term memory in mice [278]. Surprisingly, CB1 density decreased in the caffeinated mice, measured by [3H]SR141716A binding. Cortical and hippocampal tissues also showed a decrease in WIN55,212-2-stimulated [35S]GTPγS binding, but this attenuation was not seen in THC-stimulated [35S]GTPγS binding. This highlights the fact that caffeine-induced changes observed in vitro do not necessarily reflect the effects of caffeine upon integrated brain circuitry in vivo. Lastly, acute antagonism of A1 with DPCPX did not modulate the effects of THC on short-term memory [278], which further supports our hypothesis that chronic and acute blockade of A1 receptors have different functional consequences.\n\nCannabis\nCannabis and cannabis products are complex polypharmaceuticals, consisting of THC, cannabidiol (CBD), dozens of minor cannabinoids, as well as terpenoids, flavonoids, and other compounds. Fundamentally, THC mimics AEA and 2-AG by acting as an agonist at CB1 and CB2 [279]. But rather than simply substituting for AEA and 2-AG, McPartland and Guy [280] proposed that Cannabis and its many constituents work, in part, by “kick-starting” the eCB system. The acute administration of THC increased CB1 density in rodent brains [281], [282]. Acute upregulation of CB1 mRNA continued for up to 14 days in some rat brain regions [283]. Acute THC also increased the sensitivity of CB1 to cannabinoids, measured by WIN-55,212-2-stimulated [35S]GTPγS binding in rat brains [284]. Lastly, acute THC stimulated AEA biosynthesis [285].\nChronic, high dosing of THC causes a predictable desensitization and downregulation of CB1 and CB2, accompanied by drug tolerance. Chronic THC decreased CB1 density in rodent brains, and dampened cannabinoid-stimulated [35S]GTPγS [282], [284], [286], [287]. CB1 in different regions of the brain downregulate and desensitize at unequal rates and magnitudes, with greatest decreases in the hippocampus and little or no change in the nucleus accumbens and basolateral amygdala. Chronic THC elicited few changes in AEA or 2-AG levels in rat brains, except for a significant augmentation of AEA levels in the limbic forebrain [288].\nSimilar results have been reported in two human studies. Villares [289] collected postmortem brain tissues from known cannabis smokers; [3H]SR141716A binding and CB1 mRNA was downregulated in several brain regions, compared to non-smoking control autopsies. Hirvonen et al. [290] employed PET scan imaging in living subjects. The degree of CB1 downregulation correlated with years of chronic cannabis smoking. CB1 densities returned to normal after four weeks of abstinence. Variable downregulation in different brain regions may explain why frequent users of cannabis develop tolerance to some effects of THC, such as anxiogenesis and cognitive impairment, but not to its euphoric effects [291]. Downregulation is partially epigenetic—the CB1 promoter region in chronic marijuana smokers is hypermethylated, reducing CB1 mRNA expression levels [292].\nTHC acts as a partial agonist of CB1, compared to synthetic cannabinoids which act as full agonists (Table 5). Partial agonism likely explains why exposure to THC caused half as much CB1 desensitization as the full agonist WIN55,212-2 in rat hippocampal neurons [293]. In a study of rat CB1 transfected into AtT20 cells, THC caused less downregulation and internalization than WIN55,212-2 or CP-55,940 [294]. In agreement, drug tolerance studies utilizing the behavioral “tetrad” test show that chronic THC caused less tolerance than the full agonist CP-55,940 in mice [295]. In a study of human CB1 transfected into Xenopus oocytes, the desensitization rate of THC was half that of WIN55,212-2 [296]. However, one [35S]GTPγS autoradiography study of rat brains suggested that chronic THC and WIN55,212-2 caused equal desensitization [297]. Another study indicated that THC acts as a full agonist at mouse GABAergic synapses, with efficacy equal to WIN55,212-2, albeit at fairly high concentrations [298].\n10.1371/journal.pone.0089566.t005 Table 5 Partial agonism of THC at CB1, based on assays of cannabinoid-stimulated signal transduction. If THC is a partial agonist, then THC might functionally antagonize the effects of a full agonist when the two drugs are added together. THC antagonized the effects of WIN55,212-2 in rat brain sections [284], [299], and mouse autaptic hippocampal neurons [300].\nThe capacity of THC to antagonize a full agonist depends, in part, upon ligand affinity—its ability to occupy and hold the CB1 binding site. A meta-analysis of affinity studies calculated a mean Ki = 42.6 nM for THC in rat membranes—much less affinity than that of WIN-55,940, with a Kd = 2.4 nM [22]. This indicates that high concentrations of THC relative to WIN-55,940 are required to antagonize the full agonist. There are species differences—in human membranes, CB1 affinity of THC (Ki = 25.1 nM) is much closer to that of WIN-55,940 (Kd = 16.7).\n2-AG acts as a full agonist at rodent and human CB1 and CB2 [296], [301]–[303]. The emetogenic effects of exogenously-administered 2-AG were blocked by THC [304]. THC dampened or occluded eCB-mediated retrograde signaling of CB1, presumable mediated by 2-AG [300], [305], [306]. Roloff and Thayer [307] demonstrated another complexity in the relationship between THC and 2-AG: neuron firing rate in response to stimulus in rat hippocampal neurons. At low firing rates, THC mimicked 2-AG and behaved like an agonist; at high firing rates, THC antagonized endogenous 2-AG signaling.\nAEA is a partial agonist like THC, with an efficacy somewhat greater than THC in mouse brain [308] and transfected human CB1 [296]. Consistent with partial agonism, exogenously-administered AEA caused little tolerance in rodents [309], [310]. Agonist trafficking adds further complexity—THC and AEA preferentially activate different G-protein subtypes [311]. At transfected human CB1, AEA acted as a full agonist via Gαi subunits, and a partial agonist via Gαo subunits, with agonist efficacy much greater than THC at Gαi, and slightly greater than THC at Gαo [312].\nAEA and THC can antagonize each other; this in part is due to cross-tolerance [313], [314]. Falenski et al. [287] demonstrated that subchronic administration of THC in FAAH−/− knockout mice caused greater tolerance to THC than did subchronic administration of THC in wildtype mice. Thus elevated levels of AEA in FAAH−/− knockouts produced additive effects with THC. Vann et al. [315] trained rats to discriminate THC; trained rats injected with PMSF, which inhibits FAAH, showed 2.7-fold greater discrimination than rats injected with vehicle. In other words, inhibiting AEA degradation led to an increase in the potency of THC. Further, THC was more potent at producing antinociception, decreasing spontaneous activity, and increasing ring immobility when co-administered with PMSF as compared to vehicle.\nIn summary, the effects of THC upon the eCB system oscillate between potentiation and suppression, depending on acute versus chronic dosage. The dividing line between “acute” and “chronic” is a gray zone, and likely differs amongst individuals. Suplita et al. [316] summarized the situation: they studied “stress antinociception,” where rodents become less responsive to painful stimuli following exposure to an environmental stressor. Stress antinociception is mediated, in part, by the coordinated release of 2-AG and AEA. Acute administration of THC potentiated eCB-mediated stress antinociception. The converse was also true: animals exposed acutely to foot shock, which elicits eCB-mediated stress antinociception, became sensitized to the effects of THC. Chronic administration of THC predictably dampened stress antinociception. The converse was not true: chronic exposure to foot shock (3 min/day for 15 days) failed to dampen antinociception induced by either WIN-55,212-2 or by further footshocks.\nThe potential synergy between THC and the eCB system is analogous to the potential synergy between AEA and 2-AG: Rodent studies that combined FAAH and MAGL inhibitors indicated that AEA and 2-AG may activate CB1 receptors in different parts of the central nervous system. Each causes unique behavioral effects, and when both are enhanced, new effects emerge. Long and colleagues [317] showed that AEA and 2-AG independently dampen pain sensation, but together their effects are dramatically enhanced.\nCannabis is more than THC [318], [319]. Adding CBD to THC in mice enhanced CB1 expression in hippocampus and hypothalamus [320]. CBD increased hippocampal cell survival and neurogenesis, whereas THC had the opposite effect; the CBD response was absent in CB1 −/− knockout mice [321]. CBD inhibited the cellular uptake of AEA and its breakdown by FAAH [322], [323]. A separate systematic review regarding the effects of CBD on THC is currently underway (McPartland, unpublished). Several other non-THC cannabinoids interact with enzymes of the eCB system. For example, cannabidivarin and cannabidiolic acid are moderately potent inhibitors of DAGLα, and cannabigerol and cannabichromene are relatively potent inhibitors of anandamide cellular uptake [323]. Interestingly, cannabis extracts (“botanical drug substances,” BDS) enriched in cannabinoids, such as THC-acid BDS and CBD-BDS, were more potent than the corresponding pure compounds at inhibiting MAGL and AEA cellular uptake [323]."}